Colorectal cancer (CRC) is the third most commonly diagnosed cancer and a leading cause of cancer deaths in the US. The primary treatment is surgery, which can be curative especially in early stages of CRC. However, recurrence of the disease is a major problem, and is often fatal. Thus, development of new chemopreventive strategies for CRC remains a high priority. MicroRNAs (miRNAs) are an emerging class of therapeutic agents with significant translational potential for the treatment of cancer. Many different forms of cancer, including CRC, are associated with loss or reduced accumulation of one or more miRNAs that function as tumor suppressors. In animal models, restoration of the missing tumor suppressor miRNA prevents the initiation, progression and/or spread of the disease, suggesting a promising therapeutic role for these small RNAs. However, the current absence of an efficient method for systemic delivery of therapeutic miRNAs is a critical barrier to their use in cancer therapies. The proposed pilot project will test a novel chemopreventive strategy for miRNA replacement therapy based on ingestion of plants that have been bioengineered to produce therapeutic miRNAs. The work builds on our promising preliminary results showing that oral administration of plant RNA spiked with a cocktail of three tumor-suppressor miRNAs (miR-34a, -143, and -145), synthesized with the 3'-methylation characteristic of plant miRNAs, has significant chemopreventive activity in the ApcMin/+ mouse model of CRC. In Aim 1 of the proposed feasibility study, we will determine the combination of the three miRNAs that is most effective in suppressing tumorigenesis when delivered orally to ApcMin/+ mice. The research design is to feed plant RNA spiked with various combinations of synthesized tumor suppressor miRNAs to the mice either before or after tumors have developed (preventive and therapeutic regimens, respectively) and determine the impact of the treatment by assaying tumor burden, level of administered therapeutic miRNAs and expression of select miRNA target genes in treatment versus control tissues. Although the commercially synthesized miRNAs used in our pilot study and in Aim 1 allow rapid testing of specific miRNA replacement strategies, they are prohibitively expensive for translation to humans. Therefore, in Aim 2, we will establish transgenic plant line(s) producing high levels of the most effective combination(s) of tumor suppressor miRNAs as determined in Aim 1, and assay their impact when fed to ApcMin/+ mice via a custom diet. The concept of producing therapeutic miRNAs in edible plants has significant potential in basic, translational and clinical applications and provides a cost-effective alternative to currently available synthetic RNA production methods. Our group is uniquely positioned to undertake this project based on complementary expertise of the investigators. Dr. Vance is a pioneer in small RNA biology in plants and is primary co-inventor on patented technology for the use of genetically engineered miRNAs produced in plants, and Dr. Pena has many years of experience and a strong record of achievement in CRC research using mouse model systems.